Total three soil samples that were collected from Kampung Orang Asli Sungai Lalang: loamy and sandy soil. The soil samples collected from different locations were between 200–300 grams. The loamy soil consists of the combination of sandy particles, silt, and clay soil. Loamy soil conditions revealed high STHs egg density as previous reported by Nisha et al. [9] and it provided favourable conditions for the STHs growth. This is because the STHs eggs need warm and moist soil and the temperature should be over 18 °C to inhabit.
On the other hand, sandy soil type showed the absence of Ascaris eggs. This can be due to the sandy soil lack of characteristics features needed for the Ascaris eggs to fertilize. The sandy soil composed of high proportion of sand and a little clay inside. These elements did not support embryonation of Ascaris eggs, hence Ascaris eggs were not be seen in a sandy type soil. The soil samples were kept inside the refrigerator for a few days before it was being used for the isolation technique. The soil samples can tend to become dried after if it is kept for too long, the samples need to fresh for better yield. For the optimization technique, about 5 to 10 grams were kept in the incubator and incubated for 37 °C. The soil sample with viable eggs lasted up to one month in the incubator in moist conditions (distilled water was added at regular intervals to avoid dryness).
For the isolation of egg, floatation technique was used using high density floatation fluid in combination of salt/sugar solution. However, it was very difficult to get a very clear viewed of Ascaris eggs when observed under microscope as the soil samples contained a lot of debris and some small particles, despite sieving the soil samples few times prior to experiment.
The glass petri dishes were incubated for 37 °C and the embryonation stage were observed daily starting with the next day of the incubation. Subsequently, to avoid the dryness effect such as the solutions in the petri dishes was dried, also to maintain the humid environment for the eggs to develop, 2 to 3 ml of distilled water was added into the glass petri dishes. This technique was supported by the previous research by Bessat and Dewair in 2019[1]. Only distilled water was added to maintain moisture in the petri dishes containing the Ascaris eggs and the addition of sulphuric acid should be avoided since it can lead to hyper acidity affecting the developmental process.
All the stages for developmental process of Ascaris eggs can managed to be discovered completely on the 28th day. Each stage was observed for at least 2 days before the cells began to develop into more specialized form which at the end, the cell turned into larvae. From the previous research by Cruz et al. [4], each of the cell stage took at least 3 days to be observed except for the 3-cell stage. Temperature could be one of the factors of the viability of the eggs, from the previous study had mentioned that it could speed up the development of embryo if the temperature was higher compare to low temperature [4]. The first cell-stage of the eggs was observed between day two and day five. The morphology of the eggs observed was corticated layer, the thick chitin shell and undeveloped embryo of the eggs. The corticated layer was a layer that surrounded the eggs. Next, for the second cell stage, the cells began to develop, and the cleavage could be seen in this stage. The second cell stage was observed between day ten and fifteen and the eggs could be possibly turned into cleavage as early as day eight but this could be overlooked because the eggs were observed at the same time under microscope. Subsequently, the decorticated eggs that undergo the embryonation could be seen at day eighteen and finally the eggs had completed the embryonation period. The larvae development was seen in the last stage on day twenty-eight. The larvae was nicely captured and observed under microscope.
Apart from that, some of the eggs that were being cultivated by using sulphuric acid were incapable to develop as early as stage 2. This can be due to some errors during cultivating process for instance, the eggs were too fragile that the eggshells broke probably during isolating process. Next, the eggs could be dead while cultivated because the ratio of acid was too high. The amount of the sulphuric acid volumes were optimized and the other eggs managed to survive with the right proportion of sulphuric acid that suitable for aiding in cultivating process. Furthermore, the distilled water that was mixed together with the sulphuric acid during cultivating process could be contaminated and cause the eggs to not be able to develop. Nonetheless, the errors were managed to overcome successfully.